Recent studies have demonstrated that primary human fibroblasts can be directly reprogrammed to a pluripotent state upon the retroviral-mediated addition of four transgenes. The three studies presented in this thesis have sought to extend this finding to provide therapeutically relevant resources for amyotrophic lateral sclerosis (ALS) research and expand our understanding of the underlying mechanism of this direct reprogramming. In the first study, we demonstrate that these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease by generating them from two sisters -- ages 82 and 89 -- afflicted with familial ALS. We further show that these patient-specific iPS cells can be directed to differentiate into motor neurons, the cell type destroyed in ALS. These results provide for the large-scale production of disease-relevant cells directly from affected patients, a valuable substrate for disease modeling, drug discovery, and eventually autologous cell-replacement therapies. However, these initial lines may be of limited utility to the study and treatment of ALS as the vast majority of ALS cases are sporadic with no known genetic correlation. In the second study, we report the generation of a bank of 51 iPS cell lines from 15 individuals, including sporadic and familial ALS cases as well as healthy controls. In addition to providing a powerful resource for the study, and potential treatment, of this debilitating disease, the collection of cell lines reported here will also provide for a more comprehensive study of the quality and consistency of iPS cells. Finally, in a third study, we turn our attention to the mechanisms of nuclear reprogramming itself, taking two approaches to this investigation. First, we demonstrate that human fibroblasts can be partially reprogrammed by introduction of two of the iPS-generating factors. Characterization of this partially reprogrammed state suggests a decoupling of the two hallmark stem cell properties: self-renewal and pluripotency. Second, a time series during iPS induction reveals molecular milestones involved in this process and illustrates that changing the number of factors used to produce iPS cells has a profound effect on the kinetics of the process while maintaining the order of these milestone events. Taken together, the data presented in this thesis provide an initial perspective on the processes underlying direct reprogramming and achieve two key goals of regenerative medicine: the establishment of pluripotent stem cells directly from patients and their directed differentiation into cell types relevant to the patient's disease. These accomplishments will play an essential role in laying the foundation for the burgeoning field of personalized regenerative medicine.
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